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. 2015 Dec 21;36(48):3447-56.
doi: 10.1093/eurheartj/ehv460. Epub 2015 Sep 17.

The NADPH oxidase Nox4 has anti-atherosclerotic functions

Christoph Schürmann  1 , Flavia Rezende  1 , Christoph Kruse  1 , Yakub Yasar  1 , Oliver Löwe  1 , Christian Fork  1 , Bart van de Sluis  2 , Rolf Bremer  3 , Norbert Weissmann  4 , Ajay M Shah  5 , Hanjoong Jo  6 , Ralf P Brandes  7 , Katrin Schröder  7
Affiliations

The NADPH oxidase Nox4 has anti-atherosclerotic functions

Christoph Schürmann et al. Eur Heart J. .

Abstract

Aims: Oxidative stress is thought to be a risk for cardiovascular disease and NADPH oxidases of the Nox family are important producers of reactive oxygen species. Within the Nox family, the NADPH oxidase Nox4 has a unique position as it is constitutively active and produces H2O2 rather than [Formula: see text] . Nox4 is therefore incapable of scavenging NO and its low constitutive H2O2 production might even be beneficial. We hypothesized that Nox4 acts as an endogenous anti-atherosclerotic enzyme.

Methods and results: Tamoxifen-induced Nox4-knockout mice were crossed with ApoE⁻/⁻ mice and spontaneous atherosclerosis under regular chow as well as accelerated atherosclerosis in response to partial carotid artery ligation under high-fat diet were determined. Deletion of Nox4 resulted in increased atherosclerosis formation in both models. Mechanistically, pro-atherosclerotic and pro-inflammatory changes in gene expression were observed prior to plaque development. Moreover, inhibition of Nox4 or deletion of the enzyme in the endothelium but not in macrophages resulted in increased adhesion of macrophages to the endothelial surface.

Conclusions: The H2O2-producing NADPH oxidase Nox4 is an endogenous anti-atherosclerotic enzyme. Nox4 inhibitors, currently under clinical evaluation, should be carefully monitored for cardiovascular side-effects.

Keywords: ApoE; Arteriosclerosis; Inflammation; Lipids; NADPH oxidase; Reactive oxygen species; Remodelling.

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Figures

Figure 1
Figure 1
Characterization of inducible Nox4-ApoE double-knockout mice. (A) Nox4 RT-PCR from Nox4*/* and WT carotid arteries, n > 6 (left). Western blot for Nox4 and β-actin in aortae from Nox4*/* and WT mice (right). (B) Morning plasma glucose, body weight, plasma triglycerides, and cholesterol, n = 6. (C) Representative plasma lipoprotein FPLC for cholesterol and triglyceride. (D) Aortic H2O2 determined by Amplex Red/HRP from the sites indicated, n = 4. #P < 0.05 with vs. without catalase. (E) Dihydroethidium assay in arbitrary units (aU) for the O2− adduct oxyethidium (EOH) and the oxidation product ethidium (E) from aortic rings, n = 4. *P < 0.05 WT vs. Nox4*/*.
Figure 2
Figure 2
Role of Nox4 for spontaneous atherosclerosis development. (A) Photographs of oil red O staining of the aortic sinus and of the naive aorta. Scale bars are 0.5 and 20 mm, respectively. (B) Statistics of the planimetry of the vessels indicated, n ≥ 9. (C) Oil red O, Sirius red or CD68 (CD68, red and DAPI, blue) stained cross sections of brachiocephalic arteries or aortic sinus and statistics, n = 12–15 and n = 6 for CD68, Scale bars are 200 and 20 µm, respectively. *P < 0.05 WT vs. Nox4*/*. Tissue was collected at 44–49 weeks of age.
Figure 3
Figure 3
Role of Nox4 for accelerated plaque formation after partial carotid artery ligation and high-fat diet. (A) H&E cross sections of the common carotid artery of Nox4*/* mouse 28 days after partial carotid artery ligation. Arrows point to cholesterol clefts and plaque haemorrhages. (B) Volume rendering of in vivo micro-CT angiography of an Nox4*/* mouse day 14 after partial ligation of the left common carotid artery. The common carotid artery is shown and was segmented into eight equidistant parts from aorta (Aa) to bifurcation (Bf). (C–F) Side-dependent vascular lumen profile of the operated normalized to the non-operative vessels according to the segmentation denoted in (B), n ≥ 7. (G–I) Plaque volume determined by serial histological sections and time points in Nox4*/* and WT mice (G–H) and ApoE−/− mice treated with and without GKT137928 (20 mg/kg/day) (I). *P < 0.05 with vs. without GKT.
Figure 4
Figure 4
Carotid gene expression of ApoE−/− mice 7 days after ligation. Illumina MouseWG-6 v2 BeadChip array. L indicates operated left side, R indicates non-operated control side, n = 5. (A) Log2 normalized heat map of selected genes (ligated, >1.5-fold, P < 0.1) in Nox4*/* compared with WT. (B) Log2 normalized heat map of selected genes (non-ligated, >1.3-fold, P < 0.1) in Nox4*/* compared with WT. (C) Dot plot displaying relative expression level of genes in operated left (L) and control right (R) carotids of Nox4*/* and WT mice. Values are the ratios of Log10 normalized fold change.
Figure 5
Figure 5
Leucocyte adhesion on endothelial cells in vitro. (A) Adhesion of peritoneal macrophages from WT and Nox4*/* to TNFα-stimulated (10 ng/mL, 25 h) lung endothelial cells from WT mice in response to flow, n = 5–6. (B and C) Statistics and exemplary pictures of THP1 adhesion to lung endothelial cells from WT and Nox4*/* during exposure to laminar flow, n = 3. (D) Statistics of THP1 adhesion to carotid artery endothelial cells from WT and Nox4*/* during exposure laminar flow, n = 4. (E) Normalized human peripheral blood monocyte adhesion under static conditions to HUVECs pre-treated with GKT137831 (20 µM, 2 h) or solvent, n = 6. (F) HUVEC surface to total E-selectin (selE) expression determined by FACS pre-treated with GKT137831 (20 µM, 2 h) or solvent, n = 6.
Figure 6
Figure 6
Schematic on the pro-atherosclerotic effect of loss of Nox4.
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